Pneumocystis pneumonia (PCP), caused by infection with Pneumocystis jirovecii, remains an important opportunistic infection in humans. A reverse transcriptase polymerase chain reaction assay has been shown to specifically detect viable P. jirovecii organisms. In the current study, we evaluated this assay on different types of respiratory samples. The assay had a diagnostic sensitivity of 100% and a specificity of 86% when applied to bronchoalveolar lavage samples. The assay's performance declined when applied to less invasive induced sputum and oropharyngeal wash (OPW) samples. The sensitivity, when applied to OPWs, was improved by examining multiple sequential OPW samples and was affected by clinical sampling parameters that could increase or decrease the number of potential organisms in the oropharynx. When used in conjunction with an optimized clinical sampling protocol, this assay may become a useful tool for detecting and monitoring P. jirovecii in minimally invasive clinical samples.